Interleukin-10 inhibits endotoxin-induced pro-inflammatory cytokines in microglial cell cultures

Abstract

Inflammation contributes to perinatal brain injury and can be induced by hypoxia–ischemia (HI) or exposure to infection (fetal inflammatory response). The anti-inflammatory cytokine interleukin-10 (IL10) has been shown to have neuroprotective effects following HI. To determine whether IL10 can reduce the inflammatory response to lipopolysaccharide (LPS) in microglial cell cultures, primary microglial (MG) and/or HAPI cells (new MG-like cell line) were treated with LPS (50 ng/ml) in the presence or absence of IL10 (20 ng/ml) for 0.5, 1, 4, and 8 h. TNFα, MIP-1α, and RANTES were assayed by ELISA. Chemokine receptors, CCR5, CXCR3, and CX₃CR1 (fractalkine receptor) were assayed by semiquantitative RT-PCR. We found that in MG cell cultures TNFα, MIP-1α, and RANTES release after 8-h exposure to LPS was significantly higher compared to non-exposed MG cells (P<0.001). In HAPI cell cultures similar stimulation of mRNA levels was found for TNFα, MIP-1α, CXCR3, and CX₃CR1. IL10 inhibited TNFα, MIP-1α, and RANTES release of LPS-stimulated MG cells as well as TNFα, MIP-1α, and CXCR3 mRNA expression by HAPI cells after exposure to LPS (P<0.05). In contrast to those inhibitory effects, there was no change in fractalkine, and a modest increase in CX₃CR1 mRNA levels was found in the presence of IL10. We conclude that the inflammatory response induced in microglial cells by LPS can be markedly reduced by IL10. The increase in fractalkine receptor (CX₃CR1) is also potentially protective. Our results suggest that treatment of damaging neuroinflammatory insults such hypoxia–ischemia, with IL10 may be protective for the immature brain.

Introduction

Microglial (MG) cells are a major glial component of the central nervous system (CNS). They play a critical role as resident immunocompetent and phagocytic cells in the CNS (Kreutzberg, 1996), and serve as scavenger cells in the event of infection, inflammation, trauma, ischemia, and neurodegeneration in the CNS (El Khoury et al., 1998). Evidence supporting the involvement of inflammatory cytokines and chemokines in CNS injury is well documented (Liu and Ju, 1994, Liu et al., 1994, Yamasaki et al., 1995, Iadecola and Ross, 1997, Uno et al., 1997, Malek-Ahmadi, 1998, Floyd et al., 1999). Peripheral administration of LPS upregulated in brain pro-inflammatory chemokine and cytokine levels in vivo (Breder et al., 1994, Buttini and Boddeke, 1995, Quan et al., 1997, Quan et al., 1998, Vallieres and Rivest, 1997, Nadeau and Rivest, 1999a, Nadeau and Rivest, 1999b). Similar findings result from treating MG cell cultures with endotoxin (Kremlev et al., 2004).

Activation of the innate immune response in microglia is the first line of defense against invading microbials (Lehnardt et al., 2002). Microglia possess receptors that bind to the microbial derived molecular motifs. LPS is a bacterial cell wall product of gram-negative bacteria and is widely used to simulate infection under experimental conditions. LPS requires the toll-like receptor which is found on MG cells (Lehnardt et al., 2002). Microglia are the only glial cells to possess this receptor (Lehnardt et al., 2002, Lehnardt et al., 2003).

Activated microglia release soluble mediators, including oxygen and nitrogen derived free radicals, that kill developing oligodendrocytes (Haynes et al., 2003). Accordingly, methods for suppressing the inflammatory response of microglia to LPS could be neuroprotective for a wide range of neurodegenerative diseases involving activated microglia. The most likely patient to benefit might be the premature newborn between 23 and 32 weeks gestation when the developing oligodendrocytes are particularly vulnerable to attack by free radicals (Back et al., 2001). In this study, we examined the ability of interleukin-10 (IL10) to modulate the inflammatory response of MG cells exposed to LPS.

The inflammatory response of microglia includes the release of pro-inflammatory chemokines and cytokines. However, in this paper we test the hypothesis that IL10 can induce a neuroprotective effect from microglia, as microglia are also able to release mediators that tend to suppress inflammation.

Cyto- and chemokines constitute a substantial fraction of the microglial communication and effector system, involving these cells as sources of production and targets of action. Chemokines are chemotactic cytokines acting through G-protein-coupled receptors (Locati and Murphy, 1999, Rossi and Zlotnik, 2000). Microglia can produce GROα (KC), MIP-1α, MIP-1β, MIP-2, MCP-1, RANTES, and IP-10 in response to experimental stimulation by LPS, as well as cytokines, such as TNFα and IL-1. Expression patterns under normal conditions suggest neuromodulatory or supportive chemokine effects. CXCR2, CXCR3, CXCR4, CCR3, CCR5, and CX₃CR1 are among the receptors reported for microglia in vitro and/or in vivo (Glabinski and Ransohoff, 1999a, Glabinski and Ransohoff, 1999b, Biber et al., 2001). Even though the sets are not strictly complementary, the profiles of receptor and inducible chemokine expression suggest that activated microglia could serve in further microglial recruitment. Via chemokines, microglia can also affect (even support) neurons and astroglia and exert neuroprotective effects (Nau and Bruck, 2002).

Excessive MG cyto- and chemokine production due to endotoxin stimulation could cause substantial damage, which, in turn, liberates secondary signals for additional recruitment and activation of resident and invaded cells. Above a tolerable limit, mechanisms leading to cell death and tissue destruction could spread injury and eventually result in organ failure. It is feasible that the degree of CNS damage following injury is associated with MG cell activation (Giulian and Vaca, 1993), as methods to reduce MG cell activation pharmacologically via immunomodulation have been shown to be protective (Giulian and Robertson, 1990).

IL10 is a pleiotropic Th₂ cytokine that plays a key role in the regulation of inflammatory responses and immune reactions, acting on both hematopoietic and non-hematopoietic cells. IL10 has remarkable suppressive effects on the production of pro-inflammatory cytokines by monocytes–macrophages and downregulates the expression of activating molecules on these cells and dendritic cells (de Waal Malefyt et al., 1991, Fiorentino et al., 1991). Most IL10 functions are best described outside of the CNS, but recent findings extended the role of IL10 in brain. Studies performed in adult animals or in cell cultures have shown that IL10 has neuroptective properties against glutamate-induced (Bachis et al., 2001) or hypoxic–ischemic (Dietrich et al., 1999, Grilli et al., 2000) neuronal death and against LPS- or interferon-induced oligodendrocyte cell death (Molina-Holgado et al., 2001). It was also reported that IL10 counteracts acute effects of endotoxin on cerebral metabolism, microcirculation, and oxygen tension during hypoxia–ischemia in the perinatal brain (Froen et al., 2002). The goal of the present study was to determine whether IL10 can reduce the inflammatory response to LPS in microglial cell cultures. In this report, we utilized primary rat MG cells and recently characterized HAPI cells (spontaneously occurring, immortalized rat MG cell line; a “highly aggressive proliferating cell type” (Cheepsunthorn et al., 2001a, Cheepsunthorn et al., 2001b). We demonstrated that treatment with IL10 can modulate LPS-induced cyto- and chemokine release, as well as gene expression in MG cell cultures.